Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate.
The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al. (1978) J. Biol. Chem. 253, 6946]. Dihydrofolate reductase was inactivated by diethyl pyrocarbonate; the second-order rate constant for the reaction was 29 M-1 min-1 at 0 degrees C. The difference spectrum of native and diethyl pyrocarbonate inactivated enzyme had a maximum near 242 nm, which indicated a reaction with histidine residues. The absence of any spectral difference near 280 nm indicated that diethyl pyrocarbonate had not reacted with tyrosine residues. Dihydrofolate reductase lost all of its enzymatic activity after about six of the seven histidine residues had been modified. No catalytic activity was lost during an initial rapid reaction with about four histidine residues, but a subsequent slower reaction involving an additional one or two residues was associated with the loss of activity. The enzyme was protected from inactivation by either of the substrates NADPH or dihydrofolate. In fact, treatment with diethyl pyrocarbonate in the presence of either substrate, but particularly with NADPH, resulted in substantially greater activity than that found with untreated enzyme. Treatment with 1 M hydroxylamine partially restored activity to dihydrofolate reductase that had been inactivated by diethyl pyrocarbonate.
D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005493:Folic Acid Antagonists; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D006898:Hydroxylamines / Q000494:pharmacology; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology
D005493:Folic Acid Antagonists; D005561:Formates; D006898:Hydroxylamines; C010920:dihydrofolate; D006639:Histidine; D009249:NADP; D005492:Folic Acid; D004047:Diethyl Pyrocarbonate